Competitive binding immunoassays, which take advantage of natural immunological reactions, have found widespread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes which are present in very low concentration and cannot be adequately quantitated by chemical techniques. Such analytes (called ligands herein) include, for example, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc. Several techniques have been devised for determining very low concentrations of ligands. For instance, a ligand may be labeled by various means to make it readily measurable. In competitive binding assays, a labeled ligand analog (identified as ligand analog herein) is placed in competition with unlabeled ligand for reaction with a fixed amount of the appropriate binding material (called a receptor herein). Unknown concentrations of the ligand can be determined from the measured signal of either the bound or unbound (i.e. free) ligand analog. The reaction proceeds as follows:
ligand+ligand analog+receptor.revreaction. PA1 ligand-receptor+ligand analog-receptor.
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Sensitivity is of prime importance due to the extremely low level of ligands to be measured. The first highly sensitive assays used radioactive isotopes as labels. Fluorescent or enzyme labels are currently preferred in most commercial immunoassays.
Competitive binding immunoassays can also be classified as either heterogeneous or homogeneous. Heterogeneous immunoassays require a separation of bound ligand analog from free ligand analog. This separation is necessary because the properties of bound and free analog are not significantly different. Homogeneous immunoassays do not require a separation step because the properties of the bound and free analogs are different enough so that they can be differentiated.
In PCI Publication No. 82/2601 (published Aug. 5, 1982 and assigned to American Hospital Supply Corp.), a heterogeneous immunoassay carried out on a single-layer fibrous medium is described. The described assay is carried out by immunologically precipitating and immobilizing a binding material (i.e. receptor) in a finite zone of the medium, contacting the finite zone with a sample containing the ligand and a labeled indicator, washing unreacted labeled indicator out radially from the finite zone with a stream of solvent, and measuring the amount of bound labeled indicator remaining in the finite zone.
The American Hospital immunoassay requires a separate wash step to wash the free labeled indicator horizontally away from the bound labeled indicator. It would be desirable to have an immunoassay which requires no separate step for separation of bound and free ligand analog, and is therefore simpler to use and automate.
European Patent Applications Nos. 51,183 and 66,648 (both of Fuji Photo, published May 12, 1982 and Dec. 15, 1982, respectively) describe dry multilayer, analytical elements useful for heterogeneous immunoassays. These elements comprise at least three layers, including a fibrous spreading layer, a registration layer and a radiation-blocking layer between the other layers to screen out hemoglobin or other colored whole blood components. The assay is carried out by adding a test sample to the fibrous spreading layer which has a porosity suitable to allow the applied fluid to carry unbound ligand analog to the registration layer below for reaction and spectrophotometric measurement. That is, the immunoassay carried out with this element utilizes a vertical separation of bound and free ligand analog.
U.S. Pat. No. 4,258,001 (issued Mar. 24, 1981 to Pierce et al) describes a dry multilayer analytical element containing beaded spreading layers useful for a number of assays, including immunoassays. The described element comprises a particulate porous spreading layer, a reagent layer and optionally a scavenger layer to keep hydrogen peroxide out of the spreading layer (see Col. 32). In the described assay, unbound ligand analog migrates vertically from the spreading layer into the reagent layer below after the test sample is applied to the spreading layer. This reference therefore teaches a vertical separation of bound and free ligand analog.